Method of detecting auto-antibodies from patients suffering from rheumatoid arthritis, a peptide and an assay kit

ABSTRACT

Peptides useful in determining the presence of autoantibodies in patients suffering from rheumatoid arthritis are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of copending U.S. Ser. No. 13/920,017filed 17 Jun. 2013, now allowed, which is a divisional of copending U.S.Ser. No. 13/012,739 filed 24 Jan. 2011, U.S. Pat. No. 8,481,332, whichis a continuation of copending U.S. Ser. No. 10/497,667 having aninternational filing date of 11 Dec. 2002, now U.S. Pat. No. 7,888,133,issued 15 Feb. 2011, which is the national phase of PCT ApplicationPCT/NL02/00815 having an international filing date of 11 Dec. 2002, andclaims priority from Netherlands Application No. 1019540 filed 11 Dec.2001. The contents of these documents are incorporated herein byreference.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 595402000211SeqList.txt,date recorded: May 13, 2014, size: 58,196 bytes).

FIELD OF THE INVENTION

The present invention relates to a method of detecting autoantibodiesfrom patients suffering from rheumatoid arthritis, using a peptidecomprising a citrulline residue or analogue thereof.

BACKGROUND ART

Such a method is known from the international patent applicationPCT/NL97/00624. This publication describes the use of peptides derivedfrom filaggrin, and which comprise citrulline or an analogue thereof forthe detection of autoantibodies from patients suffering from rheumatoidarthritis. The peptides used are therefore suitable for diagnosticapplications, and compared with up to then, make a more reliabledetection possible. More in particular this concerns a decrease infalse-positives, i.e., a higher specificity. In addition, thesensitivity is relatively high. One peptide, indicated as cfc1, wasrecognized by 36% of the sera from patients suffering from rheumatoidarthritis. The cyclic variant of the peptide appeared to be recognizedeven better (63%).

A method according to the preamble is also known from the internationalpatent application PCT/FR00/01857. This publication describes the use ofpeptides derived from fibrin or fibrinogen, and which comprisecitrulline or an analogue thereof for the detection of autoantibodiesfrom patients suffering from rheumatoid arthritis.

PCT/EP98/07714 describes the use of synthetic peptides derived from(pro)filaggrin for the diagnosis of rheumatoid arthritis. Thisapplication also describes synthetic peptides derived from humanvimentin, cytokeratin 1, cytokeratin 9 and other intermediary filamentproteins.

However, the above-mentioned methods still only detect a limited numberof all the patients suffering from rheumatoid arthritis. Therefore thereis a strong need for a method by which an increased sensitivity can beachieved wile maintaining a substantially equal or even improvedspecificity.

DISCLOSURE OF THE INVENTION

In order to achieve this objective, the present invention provides amethod as mentioned in the preamble, which method is characterized inthat said autoantibodies are contacted with a peptide unit comprisingthe motif XG, and a peptide unit comprising the motif XnonG, wherein Xis a citrulline residue or an analogue thereof, G is the amino acidglycine and nonG is an amino acid other than glycine.

The experiments described below have shown that sera obtained frompatients suffering from rheumatoid arthritis contain two differentpopulations of antibodies. The one population is reactive with XGpeptide units such as have been described in the above-mentionedpre-published literature. The population can in part be detected withthe pre-published XG peptide units and for a still larger part whenusing the peptide units according to the present invention. The otherpopulation of antibodies reacts with XnonG peptide units. Until now,this population of antibodies has not yet been observed as such in thepre-published literature. As described below in more detail, themajority of sera from patients suffering from rheumatoid arthritiscomprise both populations of antibodies. Consequently these sera can bedetected by means of a diagnostic test comprising an XG or an XnonGpeptide unit. It appears however, that a significant part of the serafrom patients suffering from rheumatoid arthritis comprises only one ofthe two populations. Therefore, sera that comprise antibodies to theXnonG peptide unit only are not or only for a very small part detectedwith the diagnostic tests as described in the above-mentioned publishedliterature. A large part of these sera can now be detected if thediagnostic test comprises a XnonG peptide unit. An improved diagnosticmethod according to the present invention therefore comprises at leastone XG and one XnonG peptide. Thus the diagnostic test according to theinvention may be 20% more sensitive than a test according to thepublished literature.

Particularly good results were obtained when the peptide unit with theXnonG motif comprised a part not derived from natural proteins such ashuman (pro)filaggrin, fibrin, or fibrinogen as well as the relatedproteins vimentin, and cytokeratin 1 and cytokeratin 9 or other relatedintermediary filament proteins.

Surprisingly it was shown, that when combining such XnonG peptides notderived from natural proteins with XG peptides according to theinvention, a further improved diagnostic method was obtained. More inparticular, the use of such a combination of peptide units in the methodaccording to the present invention provides a diagnostic method of avery great sensitivity, while maintaining an excellent specificity. Thisis all the more surprising, since the above-mentioned publishedliterature is still based on the idea that autoantibodies from patientswith RA reacted especially well with peptides derived fromnaturally-occurring protein such a (pro)filaggrin, fibrin, fibrinogen,vimentine, cytokeratin 1 or cytokeratin 9. PCT/EP98/07714, for example,describes a filaggrin-derived XnonG peptide (IPG1249). It iscross-reactive with 3.3% of the sera from SLE patients and has ahomology of 82% with filaggrin.

Good results are obtained when the peptide unit with the XnonG motifcomprises a tripeptide, in which the central amino acid is citrulline oran analogue thereof, and that a selection is preferably made from XXK,XXY, KXI, MXR, RXY, WXK, MXH, VXK, NXR, WXS, RXW, YXM, IXX, XXF, RXH,TXV, PXH, AXF, FXR, YXF, LXM, LXY, YXP, HXS and PXW.

Preferably nonG is an amino acid selected from H, I, W, S, R, K, Y, M,F, V, P, Cit or an analogue thereof. As shown in the experiments below,such XnonG peptide units are very effective.

The peptide unit comprising the XG motif, may or may not be derived from(pro)filaggrin, fibrin, fibrinogen, vimentin, cytokeratin 1 orcytokeratin 9, and effectively the cfc1 known from PCT/NL97/00624.

In this context the term amino acid includes both natural andnon-natural amino acids, as well as amino acids having a D-configurationor L-configuration.

In the present application a non-natural amino acid is understood to bean amino acid of the kind occurring in an retro-inverso peptide,retro-peptide, a peptide wherein the side chain is located on the amidenitrogen atom of the peptide linkage, and a peptide wherein a CO of thepeptide linkage is replaced by 2, 3 or preferably a single —CH₂— group(pseudo-peptide).

Amino acids may also be modified. For example, carboxylic acid groupsmay be esterified or may be converted to an amide, and an aminogroup maybe alkylated, for example methylated. Alternatively, functional groupson the peptide may be provided with a protective group or a label (forexample a fluorescent or radioactive label). Aminogroups and carboxylicacid groups in the peptides may be present in the form of a salt formedby using an acid or a base. If synthetic peptides are used, it is verysimple to make all kinds of variants falling within the scope of theinvention that can be used. For example, aromatic side groups such asfrom phenylalanine and tyrosine may be halogenated with one or morehalogen atoms. Peptidomimetic and organomimetic embodiments also fallwithin the scope of the invention and their application in the methodaccording to the invention. Instead of a Cit residue it is also possibleto use an analogue thereof, such as those represented in FIG. 1 in theform of the amino acid. Such analogues and their preparation are knownto the person skilled in the art. For example, Sonke, et al., inStereoselective Biocatalysis (2000), pp. 23-58, and Greene: ProtectiveGroups in Organic Synthesis (Wiley, New York 1999. In accordance with afavourable embodiment, the side chains of the citrulline analogues havethe formula (I):

wherein

-   -   Q=NH₂, CH₃, NHCH₃ or N(CH₃)₂;    -   Y=O, NH, or NCH₃;    -   Z=O, NH or CH₂; and    -   n=2, 3 or 4;    -   on the condition that if Q=NH₂ and Z=NH, Y is not NH.

In this context, a peptide unit is understood to be a peptide that is atleast 7 amino acids long. Peptide units may have one or more sidechains. Also, peptide units or terminal ends of the peptide unit may beacetylated, glycosylated, alkylated, or phosphorylated independently ofeach other.

A peptide unit in this context is indicated as not derived from(pro)filaggrin, fibrin, fibrinogen, vimentin, cytokeratin 1 orcytokeratin 9 and other intermediary filament proteins if the homology(the similarity of the amino acid sequence) to these proteins is lessthan 80%, more preferably less than 75% such as less than 70% or 65%,and most preferably less than 60%, such as less than 55% or 50%. Thepeptide units shown in Table 5, which are not derived from(pro)filaggrin, fibrin, fibrinogen, vimentin, cytokeratin 1 andcytokeratin 9, have a homology between 15 and 45%. On examination of thehomology arginine, citrulline and analogues of citrulline are consideredto be equivalent (identical).

The peptide units comprising XG and XnonG may or may not part of thesame molecule. This will be entered into later on. For carrying out adetection assay, the peptide units may be bound to a carrier andoptionally provided with a label. The complex may be detected in anymanner well-known to the ordinary person skilled in the art. The complexmay be detected indirectly, for example in the case of a competitionassay, in which the complex itself is not labelled. The reaction withthe two peptide units may take place simultaneously or successively andin the same container (such as a well of a microtitre plate) or indifferent containers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a and 1 b show structures of various citrulline analogues.

MODES OF CARRYING OUT THE INVENTION

This application is not intended as an educational publication on how tobecome a person skilled in the art. It is therefore limited to providingsufficient information to enable the ordinary person skilled in the artto understand the invention, and to work it, and to understand the scopeof protection.

Preferably the peptide units are recognized by at least 2 of 100 randomsera from recognized RA patients. Obviously it is preferred to usepeptide units that are recognized by a considerably higher percentage,preferably at least 30%. The number of peptide units in a methodaccording to the invention is preferably 2 or greater than 2.

Preferably the invention relates to a method as mentioned above, whereinthe peptide unit with the XG motif and peptide unit with the XnonG motifare recognized together by at least 10% of the series of sera frompatients. Of course, combinations of peptide units resulting in highersensitivities are preferred. With some combinations of peptide units asdescribed in this document, diagnostic tests can be carried out withsensitivities of 40, 60, 70, 80 or even 85 and more than 90%.

It has been shown that the sensitivity of the detection can be furtherincreased if at least one of the peptide units is a cyclic peptide unit.

The peptide unit with XG motif and the peptide unit with XnonG motif arepreferably part of a multipeptide. In the context of the presentinvention, a multipeptide is a molecule comprised of at least twoantigenic peptide units, i.e., combinations of peptide units that may ormay not be linked by a covalent bond. Such multipeptides may becomprised of linear, branched, cyclic peptide units or a combination ofthese. Multipeptides may be comprised both of peptide units having thesame amino acid sequence, and of peptide units having different aminoacid sequences. A multipeptide according to the invention comprises atleast 7, preferably at least 10 amino acids, i.e., the peptide units mayoverlap. It goes without saying that the XG and XnonG motif can notoverlap.

The invention also relates to a XnonG peptide unit, very useful for themethod according to the present invention, comprising a sequence withthe formula (II):

(A1-A2-A3-A4-A5)-Cit-(A6)-(A7-A8-A9-A10-A11)  (II)

wherein

-   -   A1-A2-A3-A4-A5 is an amino acid sequence selected from

RHGRQ (SEQ ID NO: 13) IRCitYK (SEQ ID NO: 14) HGRQCit (SEQ ID NO: 15)GRQCitCit (SEQ ID NO: 16) FQMCitH (SEQ ID NO: 17) CitWRGM(SEQ ID NO: 18) ARFQM (SEQ ID NO: 19) QCitYKW (SEQ ID NO: 20) KPYTV(SEQ ID NO: 21) RNLRL (SEQ ID NO: 22) RRRCitY (SEQ ID NO: 23) RFKSN(SEQ ID NO: 24) RGKSN (SEQ ID NO: 25) RWVSQ (SEQ ID NO: 26) MKPRY(SEQ ID NO: 27) KSFVW (SEQ ID NO: 28) YSFVW (SEQ ID NO: 29) FQMRH(SEQ ID NO: 30) RNMNR (SEQ ID NO: 31) RMGRP (SEQ ID NO: 32)

and homologous sequences thereof;

-   -   A7 an amino acid other than glycine;    -   A7-A8-A9-A10-A11 an amino acid sequence selected from

KYIIY (SEQ ID NO: 33) TNRKF (SEQ ID NO: 34) KWCitKI (SEQ ID NO: 35)CitRAVI (SEQ ID NO: 36) RCitGHS (SEQ ID NO: 37) CitGRSR (SEQ ID NO: 38)CitYIIY (SEQ ID NO: 39) CitRLIR (SEQ ID NO: 40) IERKR (SEQ ID NO: 41)FMRKP (SEQ ID NO: 42) FMRRP (SEQ ID NO: 43) ERNHA (SEQ ID NO: 44) AVITA(SEQ ID NO: 45) TPNRW (SEQ ID NO: 46) TYNRW (SEQ ID NO: 47) RTPTR(SEQ ID NO: 48) RIVVV (SEQ ID NO: 49) HARPR (SEQ ID NO: 50) RGMCitR(SEQ ID NO: 51) IRFPV (SEQ ID NO: 52)

and homologous sequences thereof;

as well as functional analogues of the peptide with the formula (II).

The invention also relates to a XnonG peptide unit, very useful for themethod according to the present invention, comprising a sequence withthe formula (III):

(B1-B2-B3-B4-B5-B6)-Cit-(B7)-(B8-B9-B10-B11)  (III)

wherein

-   -   B1-B2-B3-B4-B5-B6 is an amino acid sequence selected from

INCitRAS (SEQ ID NO: 53) ICitKRLY (SEQ ID NO: 54) KCitCitYNI(SEQ ID NO: 55) RLYFICit (SEQ ID NO: 56) IRQGAR (SEQ ID NO: 57)CitERCitVQ (SEQ ID NO: 58) CitHQRIT (SEQ ID NO: 59) RICitRVCit(SEQ ID NO: 60) GRNQRY (SEQ ID NO: 61) RCitRQHP (SEQ ID NO: 62)CitCitRCitVA (SEQ ID NO: 63) RPKQHV (SEQ ID NO: 64) RKCitGCitR(SEQ ID NO: 65) RCitCitRNT (SEQ ID NO: 66) RCitQCitFT (SEQ ID NO: 67)QLVYLQ (SEQ ID NO: 68) QYNRFK (SEQ ID NO: 69) CitLRHIR (SEQ ID NO: 70)PRCitCitCitK (SEQ ID NO: 71) RCitQVRY (SEQ ID NO: 72) GRCitHAH(SEQ ID NO: 73) ARHVIR (SEQ ID NO: 74) RCitGHMF (SEQ ID NO: 75) GRNIRV(SEQ ID NO: 76) QIFYLCit (SEQ ID NO: 77) RQGPIA (SEQ ID NO: 78) GVYLVR(SEQ ID NO: 79) NCitCitRRV (SEQ ID NO: 80) KCitRLCitY (SEQ ID NO: 81)GRRCitCitL (SEQ ID NO: 82) RMPHCitH (SEQ ID NO: 83)

and homologous sequences thereof;

-   -   B7 is an amino acid other than glycine;    -   B8-B9-B10-B11 an amino acid sequence selected from

CitHRR (SEQ ID NO: 84) CitIRR (SEQ ID NO: 85) FRRN (SEQ ID NO: 86) AQTT(SEQ ID NO: 87) GYPK (SEQ ID NO: 88) RPPQ (SEQ ID NO: 89) GCitRK(SEQ ID NO: 90) PIPR (SEQ ID NO: 91) YTIH (SEQ ID NO: 92) RIKA(SEQ ID NO: 93) CitRVR (SEQ ID NO: 94) TRRP (SEQ ID NO: 95) TIRP(SEQ ID NO: 96) IKCitR (SEQ ID NO: 97) RNVV (SEQ ID NO: 98) CitRRY(SEQ ID NO: 99) CitRPR (SEQ ID NO: 100) TRCitCit (SEQ ID NO: 101)CitCitGR (SEQ ID NO: 102) LCitRCit (SEQ ID NO: 103) RVRCit(SEQ ID NO: 104) VPRT (SEQ ID NO: 105) YCitFR (SEQ ID NO: 106) ARCitCit(SEQ ID NO: 107) RQCitR (SEQ ID NO: 108) HIRR (SEQ ID NO: 109) CitMMR(SEQ ID NO: 110) CitRICit (SEQ ID NO: 111) VRKS (SEQ ID NO: 112)PCitCitR (SEQ ID NO: 113) CitRRK (SEQ ID NO: 114)

and homologous sequences thereof;

as well as functional analogues of the peptide with the formula (III).

The term “homologous sequence”, as used in connection withA1-A2-A3-A4-A5, A7-A8-A9-A10-A11, B1-B2-B3-B4-B5-B6 and B8-B9-B10-B11,means that at most two amino acids of each amino acid sequence may bereplaced by as many other amino acids (including citrulline and/or ananalogue thereof), at most two amino acids (including or an analoguethereof) may be introduced and at most two amino acids may be absent.

The term ‘analogue’ as used in connection with the peptide of theformula (II) or (III) means that optionally

-   -   one or more amino acids may have the D-configuration,    -   one or more side chains (other than that of citrulline or an        analogue thereof) or terminal ends of the peptide may be        acetylated, glycosylated, alkylated, or phosphorylated        independently of each other, and    -   one or more amino acids may be replaced by non-natural amino        acids.

A6 and B7 (independent of each other) are preferably selected from Cit,H, I, K, R, S, W, Y, M, F, V and P.

Specific preferred peptides to be used in the method according to theinvention are characterized in that they comprise at least one peptidesequence selected from

(SEQ ID NO: 115) R H G R Q Cit Cit K Y I I Y (SEQ ID NO: 116)I R Cit Y K Cit I T N R K F (SEQ ID NO: 117)R H G R Q Cit Cit Cit Y I I Y (SEQ ID NO: 118)A R F Q M Cit H Cit R L I R (SEQ ID NO: 119) Q Cit Y K W Cit K I E R K R(SEQ ID NO: 120) K P Y T V Cit K F M R K P (SEQ ID NO: 121)K P Y T V Cit K F M R R P (SEQ ID NO: 122) R N L R L Cit R E R N H A(SEQ ID NO: 123) R R R Cit Y Cit R A V I T A (SEQ ID NO: 124)R F K S N Cit R T P N R W (SEQ ID NO: 125) R G K S N Cit R T Y N R W(SEQ ID NO: 126) R F K S N Cit R T Y N R W (SEQ ID NO: 127)R G K S N Cit R T P N R W (SEQ ID NO: 128) R W V S Q Cit R R T P T R(SEQ ID NO: 129) M K P R Y Cit R R I V V V (SEQ ID NO: 130)K S F V W Cit S H A R P R (SEQ ID NO: 131) Y S F V W Cit S H A R P R(SEQ ID NO: 132) R N M N R Cit W R G M Cit R, and (SEQ ID NO: 133)R M G R P Cit W I R F P V

as well as

(SEQ ID NO: 134) I N Cit R A S Cit K Cit H R R (SEQ ID NO: 135)I Cit K R L Y Cit M Cit I R R (SEQ ID NO: 136)K Cit Cit Y N I Cit Cit F R R N (SEQ ID NO: 137)R L Y F I Cit Cit R A Q T T (SEQ ID NO: 138) I R Q G A R Cit R G Y P K(SEQ ID NO: 139) Cit E R Cit V Q Cit R R P P Q (SEQ ID NO: 140)Cit H Q R I I Cit V G Cit R K (SEQ ID NO: 141)R I Cit R V Cit Cit T P I P R (SEQ ID NO: 142) G R N Q R Y Cit L Y T I H(SEQ ID NO: 143) R Cit R Q H P Cit H R I K A (SEQ ID NO: 144)Cit Cit R Cit V A Cit F Cit R V R (SEQ ID NO: 145)R P K Q H V Cit H T R R P (SEQ ID NO: 146)R K Cit G Cit R Cit Cit T I R P (SEQ ID NO: 147)R Cit Cit R N T Cit H I K Cit R (SEQ ID NO: 148)R Cit Q Cit F T Cit Cit R N V V (SEQ ID NO: 149)Q L V Y L Q Cit Cit Cit R R Y (SEQ ID NO: 150)Q Y N R F K Cit Cit Cit R P R (SEQ ID NO: 151)Cit L R H I R Cit Q T R Cit Cit (SEQ ID NO: 152)P R Cit Cit Cit K Cit R Cit Cit G R (SEQ ID NO: 153)R Cit Q V R Y Cit Cit L Cit R Cit (SEQ ID NO: 154)G R Cit H A H Cit P R V R Cit (SEQ ID NO: 155)A R H V I R Cit Cit V P R T (SEQ ID NO: 156)R Cit G H M F Cit V Y Cit F R (SEQ ID NO: 157)G R N I R V Cit Cit A R Cit Cit (SEQ ID NO: 158)Q I F Y L Cit Cit H R Q Cit R (SEQ ID NO: 159) R Q G P I A Cit L H I R R(SEQ ID NO: 160) G V Y L V R Cit L Cit M M R (SEQ ID NO: 161)N Cit Cit R R V Cit M Cit R I Cit (SEQ ID NO: 162)K Cit R L Cit Y Cit P V R K S (SEQ ID NO: 163)G R R Cit Cit L Cit R P Cit Cit R (SEQ ID NO: 164)R M P H Cit H Cit S Cit R R K

or an analogue thereof.

Suitable XG peptides to be used in the method according to the inventionpreferably comprise the sequence with the formula (IV)

(C1-C2)-(C3-C4-C5)-X-G-C6-(C7-C8-C9-C10)  (IV)

wherein

C1-C2 is HQ, GF, EG or GV;

C3-C4-C5 represents 3 amino acids of which at least 1, and preferably 2independently of each other are basic, aromatic or V;

C6 is equal to a basic or aromatic amino acid, or equal to A, G, E, P,V, S or Cit or analogue thereof; and

C7-C8-C9-C10 is (SEQ ID NO: 208) SRAA, (SEQ ID NO: 209) SCitAA,(SEQ ID NO: 210) RPLD, (SEQ ID NO: 211) RVVE or (SEQ ID NO: 212) PGLD;

as well as analogues of the peptide with the formula (IV).

The term ‘analogue’ as used in connection with the peptide of theformula (IV) means that optionally

one or more amino acids may have the D-configuration,

one or more side chains (other than that of citrulline or an analoguethereof) or terminal ends of the peptide independently of each other maybe acetylated, glycosylated, alkylated, or phosphorylated; and

one or more amino acids may be replaced by non-natural amino acids.

Specific examples of XG peptides suitable to be used in the methodaccording to the invention comprise a sequence selected from

(SEQ ID NO: 165) H Q R K W Cit G A S R A A (SEQ ID NO: 166)H Q H W R Cit G A S R A A (SEQ ID NO: 167) H Q F R F Cit G Cit S R A A(SEQ ID NO: 168) H Q E R R Cit G E S R A A (SEQ ID NO: 169)H Q K W R Cit G F S R A A (SEQ ID NO: 170) H Q R W K Cit G G S R A A(SEQ ID NO: 171) H Q R R T Cit G G S R A A (SEQ ID NO: 172)H Q R R G Cit G G S R A A (SEQ ID NO: 173) H Q Cit F R Cit G H S R A A(SEQ ID NO: 174) G F F S A Cit G H R P L D (SEQ ID NO: 175)H Q E R G Cit G K S R A A (SEQ ID NO: 176) H Q E K R Cit G K S R A A(SEQ ID NO: 177) H Q R W L Cit G K S R A A (SEQ ID NO: 178)H Q K R N Cit G K S R A A (SEQ ID NO: 179) E G G G V Cit G P R V V E(SEQ ID NO: 180) H Q W R H Cit G R S Cit A A (SEQ ID NO: 181)H Q K W N Cit G R S R A A (SEQ ID NO: 182) H Q K F W Cit G R S R A A(SEQ ID NO: 183) H Q K Cit K Cit G R S R A A (SEQ ID NO: 184)H Q K W R Cit G R S Cit A A (SEQ ID NO: 185) H Q A W R Cit G R S Cit A A(SEQ ID NO: 186) H Q N Q W Cit G R S R A A (SEQ ID NO: 187)H Q N S K Cit G R S R A A (SEQ ID NO: 188) H Q K R R Cit G R S R A A(SEQ ID NO: 189) H Q K R F Cit G R S R A A (SEQ ID NO: 190)H Q K R Y Cit G R S R A A (SEQ ID NO: 191) H Q K R H Cit G R S R A A(SEQ ID NO: 192) H Q E R A Cit G S S R A A (SEQ ID NO: 193)H Q E K M Cit G V S R A A (SEQ ID NO: 194) H Q K R G Cit G W S R A A(SEQ ID NO: 195) H Q R R V Cit G W S R A A (SEQ ID NO: 196)H Q W N R Cit G W S R A A (SEQ ID NO: 197) H Q Q R M Cit G W S R A A(SEQ ID NO: 198) H Q S H R Cit G W S R A A (SEQ ID NO: 199)H Q F R F Cit G W S R A A (SEQ ID NO: 200) H Q K R R Cit G W S R A A(SEQ ID NO: 201) G V K G H Cit G Y P G L D

or an analogue thereof.

The peptides according to the invention are preferably cyclic peptidesof which the ring comprises at least 10 amino acids, and more preferablyat least 11 amino acids. The person skilled in the art is acquaintedwith various methods for the preparation of cyclic peptides and afurther explanation is not required.

According to a most preferred method of the invention, an XG peptide isused in combination with at least one XnonG peptide, wherein the XGpeptide is selected from the group comprised of:

(SEQ ID NO: 1) 0002-27 H Q K R G Cit G W S R A A  (SEQ ID NO: 2)0002-29 H Q R R V Cit G W S R A A  (SEQ ID NO: 3)0002-31 H Q R R T Cit G G S R A A  (SEQ ID NO: 4)0002-32 H Q R K W Cit G A S R A A  (SEQ ID NO: 5)0002-36 H Q F R F Cit G Cit S R A A  (SEQ ID NO: 6)0002-37 H Q K W R Cit G R S Cit A A  (SEQ ID NO: 7)0002-63 H Q F R F Cit G W S R A A 

and the XnonG peptide is chosen from the group comprised of:

(SEQ ID NO: 8) 0107-32 K P Y T V Cit K F M R R P  (SEQ ID NO: 9)0107-35 A R F Q M Cit H Cit R L I R  (SEQ ID NO: 10)0107-45 Y S F V W Cit S H A R P R  (SEQ ID NO: 11)0113-30 A R F Q M R H Cit R L I R  (SEQ ID NO: 12)0218-36 R N L R L Cit R E R N H A 

Depending on the desired specificity and sensitivity of the diagnostictest and the respective population of rheumatoid arthritis sera underexamination, a preferred combination of XG and XnonG peptide units isselected from the group comprised of:

0002-27 and 0107-32; 0002-27 and 0107-35; 0002-27 and 0107-45;

0002-27 and 0113-30; 0002-27 and 0218-36; 0002-29 and 0107-32; 0002-29and 0107-35; 0002-29 and 0107-45; 0002-29 and 0113-30; 0002-29 and0218-36; 0002-31 and 0107-32; 0002-31 and 0107-35; 0002-31 and 0107-45;0002-31 and 0113-30; 0002-31 and 0218-36; 0002-32 and 0107-32; 0002-32and 0107-35; 0002-32 and 0107-45; 0002-32 and 0113-30; 0002-32 and0218-36; 0002-36 and 0107-32; 0002-36 and 0107-35; 0002-36 and 0107-45;0002-36 and 0113-30; 0002-36 and 0218-36; 0002-37 and 0107-32; 0002-37and 0107-35; 0002-37 and 0107-45; 0002-37 and 0113-30; 0002-37 and0218-36; 0002-63 and 0107-32; 0002-63 and 0107-35; 0002-63 and 0107-45;0002-63 and 0113-30; 0002-63 and 0218-36.

The above-mentioned combinations were shown to produce an average againin sensitivity of 6%, bringing the total sensitivity of such acombination test of an XG and a XnonG peptide to 75 to 78%.

The results described in the examples below further showed that thevarious peptide units also detected different cohorts of sera. Anadditional gain in sensitivity was achieved by adding a third peptideunit or even a fourth or more peptide units. Depending on thecombination of peptide units selected from the above-mentioned groups, adiagnostic test according to the invention allowed sensitivities of88-92% to be achieved.

The invention also relates to a multipeptide, characterized in that itis a linear or branched multipeptide, comprising at least two linear orcyclic peptide sequences selected independently of each other from

a peptide unit selected from peptide units with the formula (II) and(III) and analogues thereof;

a peptide unit with the formula (IV) and analogues thereof.

Such a multipeptide is very suitable for use in the method according tothe invention. It makes it possible to carry out the method more simplyand more reliably since peptides are used in the same known ratio, andextra operations during the assay or during the preliminary work (e.g.,coating a microtitre plate with multipeptide) is avoided.

The invention further relates to a diagnostic kit for determining thepresence of autoantibodies from patients suffering from rheumatoidarthritis, wherein the diagnostic kit comprises a peptide or amultipeptide according to the invention, or a mixture thereof, togetherwith at least one further reagent.

The invention also relates to a peptide or an antibody of an immunotoxinmolecule as described above, or a composition thereof for use as apharmaceutical composition.

The present invention further relates to a peptide or an antibody of animmunotoxin molecule as described above or a composition thereof forpreparing a pharmaceutical composition or a diagnostic agent forrheumatoid arthritis.

The present invention also relates to the application of a peptide or acomposition thereof as described above for preparing a pharmaceuticalcomposition for the treatment of autoimmune diseases by increasing thesize of antigen immune complexes, which improves the clarification ofthe immune complexes formed.

The present invention therefore also relates to a method for thetreatment of rheumatoid arthritis by introducing into the body of apatient requiring such treatment, at least one peptide according to theinvention.

The invention further relates to a method for the selection of a peptidesuitable for the diagnosis of RA, wherein a peptide library is screenedwith antibodies obtained from patients with RA and wherein the peptidelibrary is selected from a group comprised of:

(SEQ ID NO: 202) Lib (1): H Q E X X Cit X X S R A A

Wherein X=any amino acid except cysteine and tryptophane

(SEQ ID NOS: 203-204) Lib (2): H Q X X X Cit G X S R/Cit A A

Wherein X=any amino acid except cysteine but including Citrulline

(SEQ ID NOS: 205-206) Lib (3): H Q E X X Cit X X S R/Cit A A

Wherein X=any amino acid except cysteine but including Citrulline

(SEQ ID NO: 207) Lib (4): X X X X X X Cit X X X X X

Wherein X=any amino acid except cysteine but including citrulline

or equivalents thereof.

Finally, the invention relates to a peptide that can be obtained withthe aid of the afore-mentioned method to be used in a diagnostic assay.

The terms “a pharmaceutical composition for the treatment” or “a drugfor the treatment” or “the use of proteins for the preparation of a drugfor the treatment” relate to a composition comprising a peptide asdescribed above or an antibody binding specifically to the peptide and apharmaceutically acceptable carrier or excipient (the two terms areinterchangeable) for the treatment of diseases as described above.Suitable carriers or excipients with which the ordinary person skilledin the art is familiar are saline, Ringer's solution, dextrose solution,Hank's solution, oils, ethyl oleate, 5% dextrose in saline, substancesimproving isotonicity and chemical stability, buffers and preservatives.Other suitable carriers include any carrier that does not itself inducethe production of antibodies harmful to the individual receiving thecomposition, such as proteins, polysaccharides, polylactic acids,polyglycol acids, polymeric amino acids and amino acid polymers. The“drug” may be administered in any manner known to the ordinary personskilled in the art.

The peptides according to the invention may be labelled (radioactive,fluorescent or otherwise, as well known in the art) or may be providedwith a carrier. In this form also such peptides fall within the scope ofthe invention. For example, a peptide may be coupled to a carrierprotein, such as Keyhole Limpet Haemocyanin or bovine serum albumin.Also, the peptide according to the invention may be non-covalently orcovalently coupled to a solid carrier, such as a microsphere (gold,polystyrene, etc.), slides, chips, or a wall of a reactor vessel or of awell of a microtitre plate. The peptide may be labelled with a direct oran indirect label. Examples include biotin, fluorescein and an enzyme,such as horseradish peroxidase. All this is generally known to theordinary person skilled in the art and requires no further explanation.

EXAMPLES Example 1 Peptide Synthesis

Citrullinated peptides were synthesised as described by De Koster, H.S., et al. (J. Immunol. Methods, 187, pp. 177-188, (1995)). Beads wereused to which the peptide molecules were attached via an amide bond sothat after the removal of protective groups, peptide molecules werestill attached to the bead. For the synthesis of peptides an automatedmultiple peptide synthesiser was used (Abimed AMS422, Abimed,Langenfeld, Germany). The incorporated amino acids and the peptidelinker 6-aminohexane acid were protected by a Fmoc-group, and tofacilitate coupling, the protected amino acids were activated with PyBOPand N-methylmorpholine. Where necessary, the side chains were protectedwith groups protecting acid-sensitive side chains. The beads used had adiameter of approximately 100 μm and comprised approximately 100 pmolpeptide each. It is estimated that approximately 0.5% of this amount isbound to the outside and is in principle accessible to antibodies.

For the synthesis in larger quantities, beads were used that wereprovided with an acid-sensitive linker. The chemistry of peptidesynthesis was largely comparable with the one already described above.The peptides were split off with trifluoroacetic acid and isolated bymeans of ether precipitation, all in accordance with methods well-knownin the art and for which the ordinary person skilled in the art requiresno further explanation. It is of course also possible to acquirepeptides with a desired sequence commercially. The purity and identityof the individual peptides were checked by means of analytical RP-HPLCand time-of-flight mass spectrometry (MALDI-TOF).

With the aid of the above method 4 peptide libraries were created, eachcomprising a large number (8×10⁶) citrullinated peptides. The formulasbelow show the amino acid sequence of the peptides in each library:

(SEQ ID NO: 202) Lib (1): H Q E X X Cit X X S R A A

Wherein X=any amino acid except cysteine and tryptophane

(SEQ ID NOS: 203-204) Lib (2): H Q X X X Cit G X S R/Cit A A

Wherein X=any amino acid except cysteine but including Citrulline

(SEQ ID NOS: 205-206) Lib (3): H Q E X X Cit X X S R/Cit A A

Wherein X=any amino acid except cysteine but including Citrulline

(SEQ ID NO: 207) Lib (4): X X X X X X Cit X X X X X

Wherein X=any amino acid except cysteine but including Citrulline.

These libraries were screened in accordance with the method describedbelow using sera from patients suffering from rheumatoid arthritis.

Example 2 Reaction of the Bead-Coupled Peptides with Sera from PatientsSuffering from Rheumatoid Arthritis (RA)

With the aid of Protein A-Sepharose, IgG was isolated from serum frompatients clinically diagnosed to be suffering from RA. The beads wereincubated with a solution comprising i) total serum from the patient, orii) IgG from a patient conjugated to a reporter enzyme (alkalinephosphatase labelling kit; Roche/Boehringer, Mannheim, Germany). Forserum (i) a second incubation was carried out with an alkalinephosphatase conjugated anti-human IgG antibody (Dako D0336; DakoImmunoglobulins, Glostrup, Denmark). After each incubation the beadswere thoroughly washed with Tris-HCl buffer pH 8.9 (50 mM Tris pH 8.9,150 mM NaCl, 0.5% Tween® 20).

The beads with peptides that had bound the most human IgG (after dyingwith a substrate of alkaline phosphatase that is converted into aninsoluble coloured product, these become the most intensely colouredbeads) were selected with the aid of a microscope.

Example 3 ELISA

The most interesting peptides were synthesised in a slightly largerquantity to form a linear and a cyclic variant, and in most cases in acitrulline and arginine (=control) variant. These peptides were testedfor reactivity with a series of sera from RA patients. The peptides werecoated to ELISA plates and incubated with sera from RA patients. Eachserum was tested in duplicate. Sera from healthy persons were used asnegative control.

Example 4 The Family of XG Peptides

By using sera that gave a positive reaction with cfc1 (seePCT/NL97/00624, which is herewith included by way of reference) peptideswith glycine were found on position +1 (i.e., C-terminal) in relation tothe citrulline residue. Replacing G on +1 by, for example, A (alanine)reduces the reactivity by 65%. In certain peptides, the E (glutamicacid) on position −3 is preferably not replaced by A (47% loss ofactivity). Similarly, the Q (glutamine) on −4, the H (histidine) on −5and the S (serine) on +3 often appeared to be advantageous.

Thus a number of peptide sequences with the combination —XG— wereselected, all of which reacted with RA sera. Specific examples ofpreferred members of this XG family are

TABLE 1 Family of XG peptides:H Q R K W Cit G A S R A A (SEQ ID NO: 165) (0002-32)H Q H W R Cit G A S R A A (SEQ ID NO: 166) (0002-42)H Q F R F Cit G Cit S R A A (SEQ ID NO: 167) (0002-36)H Q E R R Cit G E S R A A (SEQ ID NO: 168) (020699-2)H Q K W R Cit G F S R A A (SEQ ID NO: 169) (0102-74)H Q R W K Cit G G S R A A (SEQ ID NO: 170) (0002-28)H Q R R T Cit G G S R A A (SEQ ID NO: 171) (0002-31)H Q R R G Cit G G S R A A (SEQ ID NO: 172) (0002-33)H Q Cit F R Cit G H S R A A (SEQ ID NO: 173) (0002-43)G F F S A Cit G H R P L D (SEQ ID NO: 174) (0101-7)H Q E R G Cit G K S R A A (SEQ ID NO: 175) (020699-1)H Q E K R Cit G K S R A A (SEQ ID NO: 176) (020699-4)H Q R W L Cit G K S R A A (SEQ ID NO: 177) (0002-30)H Q K R N Cit G K S R A A (SEQ ID NO: 178) (0002-38)E G G G V Cit G P R V V E (SEQ ID NO: 179) (0101-5)H Q W R H Cit G R S Cit A A (SEQ ID NO: 180) (0002-34)H Q K W N Cit G R S R A A (SEQ ID NO: 181) (0002-24)H Q K F W Cit G R S R A A (SEQ ID NO: 182) (0002-25)H Q K Cit K Cit G R S R A A (SEQ ID NO: 183) (0002-26)H Q K W R Cit G R S Cit A A (SEQ ID NO: 184) (0002-37)H Q A W R Cit G R S Cit A A (SEQ ID NO: 185) (0002-40)H Q N Q W Cit G R S R A A (SEQ ID NO: 186) (0002-44)H Q N S K Cit G R S R A A (SEQ ID NO: 187) (0002-45)H Q K R R Cit G R S R A A (SEQ ID NO: 188) (0102-76)H Q K R F Cit G R S R A A (SEQ ID NO: 189) (0102-77)H Q K R Y Cit G R S R A A (SEQ ID NO: 190) (0102-78)H Q K R H Cit G R S R A A (SEQ ID NO: 191) (0102-79)H Q E R A Cit G S S R A A (SEQ ID NO: 192) (020699-3)H Q E K M Cit G V S R A A (SEQ ID NO: 193) (020699-5)H Q K R G Cit G W S R A A (SEQ ID NO: 194) (0002-27)H Q R R V Cit G W S R A A (SEQ ID NO: 195) (0002-29)H Q W N R Cit G W S R A A (SEQ ID NO: 196) (0002-35)H Q Q R M Cit G W S R A A (SEQ ID NO: 197) (0002-41)H Q S H R Cit G W S R A A (SEQ ID NO: 198) (0002-39)H Q F R F Cit G W S R A A (SEQ ID NO: 199) (0002-63)H Q K R R Cit G W S R A A (SEQ ID NO: 200) (0102-75)G V K G H Cit G Y P G L D (SEQ ID NO: 201) (0101-3)

From the above data a consensus sequence is derived of amino acids thatappear to have a preference for a particular position. An XG peptideunit being preferably recognized by RA sera may therefore be representedby:

−3 −2 −1 0 +1 +2  K  R  R X  G  R  R  W          W  E             K

All these peptides showed good to excellent results. Especially thepeptides 0002-35, 0002-36 and 0002-63 were very satisfactory. In most ofthe cases the cyclic variant of such a peptide reacted even better (seelater). Incidentally, this is not always the case. For example, thelinear peptide 0101-7 reacted with 55% of the sera. The cyclic variantof this peptide reacted with “only” 45% of 186 RA-sera. As expected, thearginine variants of these peptides (wherein the citrulline(s) is/arereplaced by arginine(s)) did not react with the RA sera.

Example 5 Cyclic Variants of XG Peptides

The peptides described below were cyclisized in accordance with thecysteine-bromoacetic acid method. Several cyclic variants were tested,with in particular the cyclic variants of peptides 0002-27, 0002-29,0002-31, 0002-36, and 0002-63 proving to be of interest.

Ring size: a number of adequately reacting peptides were used tosynthesise additional cyclic peptides that each had a different ringsize. Of these the 8, 9, 10, 11 and 12-mers were eventually tested. Allthese peptides were tested in ELISA with 48 RA sera and 8 normal serarespectively. The 8- and 9-mers reacted with some of the sera (with 22%(titre 0.47) and 28% (titre 0.5), respectively, but the titres (theOD-values found) were much lower than when larger rings were used(10-mer 33% with a titre van 0.60; 11- and 12-mer 50% with an averagetitre of 0.74.

TABLE 2 Results from serum-analyses using cyclic peptide 0002-36. N %positive sera RA 318 71 Systemic Lupus 24 0 Erythematodes Prim Sjögren'ssyndrome 34 3 SSc Scleroderma 10 0 PM, Polymyositis 11 0 Osteoarthritis29 0 Crohn's disease 40 0 Colitis ulcerosa 40 0 Infectious diseases(malaria, 250 1 chlamydia, mycoplasma, etc.) Psoriasis 10 0 Vasculitis30 0 Normal human serum 84 0

The above table represents ELISAs with sera obtained from patientssuffering from RA and from patients suffering from various otherautoimmune diseases. This shows the high specificity of RA sera for apeptide such as 0002-36. Practically none of the cases showed seraobtained from other autoimmune diseases and material from normal healthypersons to be positive. The arginine variant of peptide 0002-36 (R onposition 0 and +2, i.e., instead of Cit in the formula) was also testedand shown to be negative with all tested RA and normal sera.

TABLE 3 ELISA with 132 RA sera with peptide 36 compared with peptidecfc1 from [PCT/NL97/00624]: cfc1 cfc1 0002-36 0002-36 peptide (linear)(cyclic) (linear) (cyclic) positive sera 48/132 83/132 80/132 94/132 36%63% 61% 71%

By using cyclic cfc1 as well as cyclic 0002-36 an additional 2%sensitivity may be gained (total 73%, see Table 4), which for this typeof application is a welcome improvement as long as it is not accompaniedby a declining specificity. The latter does not seem to be the case.Thus within the XG group of peptides there is diversity with regard totheir ability to react with RA antibodies.

TABLE 4 ELISA with 186 RA sera and cyclic variants of cf1, 0002-36, and0002-63 cfc1 0002-63 0002-36 cfc1 + 0002-36 peptide (cyclic) (cyclic)(cyclic) (cyclic) positive sera 51% 71% 71% 73%

Comparing the linear and the cyclic variants of 0002-36 in two cohortsof RA sera (in total 318 sera), the cyclic variant was shown to reactmore frequently (and better) than the linear variant. Better, becausethe OD-values found with the cyclic variant were also much higher thanwith the linear composition. Whether the cyclic variant was created via—S—S-bonds between two cysteins, or via a thioether formed, for example,by reacting a cysteine and a bromoacetyl group (formed by reacting athiol in the side chain of a cysteine with an N-terminal bromoacetylgroup, forming a thioether), is immaterial. The N-terminal bromoacetylgroup is formed, after the synthesis of the peptide, by allowing thebead-bound peptide (peptidyl-resin) to react with the N-hydroxysuccinimide ester of bromoacetic acid) in the same peptide. The ringclosure occurs in a phosphate-buffered aqueous acetonitril solution,pH=8.

Example 6 The family of XnonG peptides

Surprisingly, there were also peptides found that were not derived from(pro)filaggrin, fibrin, fibrinogen, vimentin, cytokeratin 1 andcytokeratin 9, and that were recognized by RA sera that did not or onlyslightly react with peptides of the XG family described above. Acharacteristic of these peptides is that the amino acid and theC-terminal side of the Cit is basic (R, K, H), aromatic (W, Y, F)aliphatic (M, I, V) or Cit, S or P (see Table 5), but not G. Hence thename XnonG family. Remarkably, XnonG peptides comprise relatively manypositively charged amino acids on the −2, −1, +1 and +2 positions. Thusthe antibodies in these RA sera react with a citrulline in a differentpeptide context.

The XnonG peptides mentioned all reacted with one or more XG-negativesera. For example, peptides 0107-32, 0107-35, and 0107-45 react withapproximately 15-18% of the XG negative sera. An ELISA test based on oneor more of these peptides induces a sensitivity increase of at least 5%(up from 73% to 78%) compared with the combination of cyclic cfc1 andcyclic 0002-36. Very importantly, none of the XnonG peptides mentionedreacted with control sera.

TABLE 5 Peptides of the XnonG family:R H G R Q Cit Cit K Y I I Y (SEQ ID NO: 115) (0107-33)R H G R Q Cit Cit Cit Y I I Y (SEQ ID NO: 116) (0107-34)I R Cit Y K Cit I T N R K F (SEQ ID NO: 117) (0107-37)A R F Q M Cit H Cit R L I R (SEQ ID NO: 118) (0107-35)Q Cit Y K W Cit K I E R K R (SEQ ID NO: 119) (0107-43)K P Y T V Cit K F M R K P (SEQ ID NO: 120) (0107-31)K P Y T V Cit K F M R R P(SEQ ID NO: 121) (0107-32)R N L R L Cit R E R N H A (SEQ ID NO: 122) (0107-36)R R R Cit Y Cit R A V I T A (SEQ ID NO: 123) (0107-38)R F K S N Cit R T P N R W (SEQ ID NO: 124) (0107-42)R G K S N Cit R T Y N R W (SEQ ID NO: 125) (0107-39)R F K S N Cit R T Y N R W (SEQ ID NO: 126) (0107-40)R G K S N Cit R T P N R W (SEQ ID NO: 127) (0107-41)R W V S Q Cit R R T P T R (SEQ ID NO: 128) (0102-71)M K P R Y Cit R R I V V V (SEQ ID NO: 129) (0102-73)K S F V W Cit S H A R P R (SEQ ID NO: 130) (0107-44)Y S F V W Cit S H A R P R (SEQ ID NO: 131) (0107-45)R N M N R Cit W R G M Cit R (SEQID NO: 132) (0107-30)R M G R P Cit W I R F P V (SEQ ID NO: 133) (0102-72) as well asI N Cit R A S Cit K Cit H R R (SEQ ID NO: 134) 0215-46I Cit K R L Y Cit M Cit I R R (SEQ ID NO: 135) 0219-44K Cit Cit Y N I Cit Cit F R R N (SEQ ID NO: 136) 0222-56R L Y F I Cit Cit R A Q T T (SEQ ID NO: 137) 0222-57I R Q G A R Cit R G Y P K (SEQ ID NO: 138) 0223-12Cit E R Cit V Q Cit R R P P Q (SEQ ID NO: 139) 150202RA02Cit H Q R I T Cit V G Cit R K (SEQ ID NO: 140) 150202RA03R I Cit R V Cit Cit T P I P R (SEQ ID NO: 141) 061102RA01G R N Q R Y Cit L Y T I H (SEQ ID NO: 142) 061102RA02R Cit R Q H P Cit H R I K A (SEQ ID NO: 143) 061102RA03Cit Cit R Cit V A Cit F Cit R V R (SEQ ID NO: 144) 061102RA05R P K Q H V Cit H T R R P (SEQ ID NO: 145) 061102RA06R K Cit G Cit R Cit Cit T I R P (SEQ ID NO: 146) 061102RA07R Cit Cit R N T Cit H I K Cit R (SEQ ID NO: 147) 061102RA08R Cit Q Cit F T Cit Cit R N V V (SEQ ID NO: 148) 061102RA09Q L V Y L Q Cit Cit Cit R R Y (SEQ ID NO: 149) 061102RA11Q Y N R F K Cit Cit Cit R P R (SEQ ID NO: 150) 061102RA12Cit L R H I R Cit Q T R Cit Cit (SEQ ID NO: 151) 061102RA14P R Cit Cit Cit K Cit R Cit Cit G R (SEQ ID NO: 152) 061102RA15R Cit Q V R Y Cit Cit L Cit R Cit (SEQ ID NO: 153) 061102RA16G R Cit H A H Cit P R V R Cit (SEQ ID NO: 154) 061102RA17A R H V I R Cit Cit V P R T (SEQ ID NO: 155) 181102RA01R Cit G H M F Cit V Y Cit F R (SEQ ID NO: 156) 181102RA02G R N I R V Cit Cit A R Cit Cit (SEQ ID NO: 157) 181102RA04Q I F Y L Cit Cit H R Q Cit R (SEQ ID NO: 158) 221102RA01R Q G P I A Cit L H I R R (SEQ ID NO: 159) 221102RA02G V Y L V R Cit L Cit M M R (SEQ ID NO: 160) 0218-36N Cit Cit R R V Cit M Cit R I Cit (SEQ ID NO: 161) 271102RA01K Cit R L Cit Y Cit P V R K S (SEQ ID NO: 162) 271102RA02G R R Cit Cit L Cit R P Cit Cit R (SEQ ID NO: 163) 271102RA03R M P H Cit H Cit S Cit R R K (SEQ ID NO: 164) 271102RA04

From the above data it is possible to derive a consensus sequence ofamino acids that appear to have a preference for certain positions.Therefore, a XnonG peptide unit preferably recognized by RA sera may berepresented by:

−5 −4 3 −2 −1 0 +1 +2 +3 +4 +5  R  R R  R  R X  R  R  R  R  R    N Y  V  Q    H  T  I  I  K       F  I  Y          Y  P  P      H  S  V             N       G

Experiments have shown that, for example, peptide 0107-35 in the cyclicform reacts with 18% of the sera that are not reactive with cyclic0002-36. This sensitivity may be further increased by other peptides.For example, a further 8% may be added by cyclic peptide 0107-32. Thisallows the sensitivity to be increased to 80%. A similar value was foundfor the peptide 0107-45. In total 17 of the 52 (32%) XG-negative serawere shown to react with one or more XnonG peptides. This means thatwith combinations of more than 2 XG and XnonG peptides, a preferredembodiment of the invention, a sensitivity of above 80% can be achieved.This is therefore better than what can be achieved by a combination ofthe peptides with the formula IV, and those known from PCT/NL97/00624.

The sensitivity can be increased even further by using more peptidesstill. When testing the RA sera with a cyclic XG peptide (for example0002-63 or 0002-36) together with a linear or cyclic XnonG peptide (forexample 0107-35), for 318 RA sera a sensitivity of 78% was obtained. Theaddition of a 3rd, 4th and possibly 5th peptide increased thesensitivity to 85% (peptides 0107-32, 0107-42 respectively 0107-34). Amere 48 of the 318 RA sera did not react with one of the peptidesmentioned.

Example 7 Sensitivity and Specificity of Diagnostic Tests Comprising XGand XnonG Peptide Units

Using the above-described methods, seven XG peptide units were testedfor reactivity with the 318 sera mentioned in Table 2 from patientssuffering from rheumatoid arthritis. The specificity was determined withthe aid of sera mentioned in Table 2 from control patients and normaldonors. Table 6 shows the specificity and sensitivity of the individualpeptide units.

TABLE 6 XG Peptide Sensitivity [%] Specificity [%] units Linear cycliclinear cyclic 0002-27 56 71 98 98 0002-29 57 70 98 98 0002-31 51 69 9898 0002-32 61 71 98 98 0002-36 61 71 99 99 0002-37 60 70 98 98 0002-6360 71 98 98 cfc1 36 56 — —

In accordance with the methods described in this document, five XnonGpeptide units were tested for reactivity with the same 318 seramentioned in Table 2 from patients suffering from rheumatoid arthritis.Specificity was again determined with the aid of the sera mentioned inTable 2 from control patients and normal donors. Table 7 shows thespecificity and sensitivity of the individual peptide units.

TABLE 7 Sensitivity [%] Specificity [%] Peptide unit Linear cycliclinear cyclic 0107-32 48 63 98 98 0107-35 52 61 98 99 0107-45 51 69 9899 0113-30 49 71 99 100 0218-36 50 70 98 98

The reactivity of the XnonG peptide units mentioned in Table 7 wastested with 80 sera from the panel of 318 sera described above that didnot react with any of the XG peptide from Table 6. The percentagesmentioned in table 8 therefore show the percentage of the sera that didcomprise antibodies to XnonG peptide units but comprised no XG

TABLE 8 Sensitivity [%] Peptide unit Linear cyclic 0107-32 15 18 0107-3517 18 0107-45 16 18 0113-30 18 19 0218-36 17 17

The above results show that an increased sensitivity may be expected ifeach of the XG peptide units from Table 6 is combined with each of thepeptide units from Table 7. All the combinations of peptide units givenbelow were tested with a representative portion of the above-mentionedpanel of 318 sera from patients suffering from rheumatoid arthritis.This experiment does in fact show that an average gain in sensitivity of6% was obtained, bringing the total sensitivity of such a combinationtest to 75 to 78%.

The tested combinations of peptide units related to: peptide unit0002-27 with 0107-32; 0002-27 with 0107-35; 0002-27 with 0107-45;0002-27 with 0113-30; 0002-27 with 0218-36; 0002-29 with 0107-32;0002-29 with 0107-35; 0002-29 with 0107-45; 0002-29 with 0113-30;0002-29 with 0218-36; 0002-31 with 0107-32; 0002-31 with 0107-35;0002-31 with 0107-45; 0002-31 with 0113-30; 0002-31 with 0218-36;0002-32 with 0107-32; 0002-32 with 0107-35; 0002-32 with 0107-45;0002-32 with 0113-30; 0002-32 with 0218-36; 0002-36 with 0107-32;0002-36 with 0107-35; 0002-36 with 0107-45; 0002-36 with 0113-30;0002-36 with 0218-36; 0002-37 with 0107-32; 0002-37 with 0107-35;0002-37 with 0107-45; 0002-37 with 0113-30; 0002-37 with 0218-36;0002-63 with 0107-32; 0002-63 with 0107-35; 0002-63 with 0107-45;0002-63 with 0113-30 and finally 0002-63 with 0218-36.

With respect to the results of Table 6 and Table 8 it should also benoted that the various peptide units also detected different cohorts ofsera. From this it may be deduced that the above-mentioned combinationsof XG and XnonG peptide units also are capable of producing a furthergain in sensitivity if a third peptide unit or even a fourth or furtherpeptide units are added. Depending on the selected combination ofpeptide units, the diagnostic test according to the invention did indeedmake it possible to achieve a sensitivity of 88-92%.

1. A method to detect the presence or absence of autoantibodies associated with rheumatoid arthritis (RA), which method comprises contacting a sample suspected of containing said antibodies with a peptide comprising the motif XG and a peptide comprising the motif XnonG, wherein X is a citrulline residue or an analog thereof, G is the amino acid glycine and nonG is an amino acid other than glycine for a time sufficiently long to allow a complex to be formed between the at least one of said peptides and the autoantibodies, and detecting the presence or absence of the complex wherein the presence of said complex detects the presence of said antibodies.
 2. The method of claim 1, wherein nonG is an amino acid selected from H, I, W, S, R, K, Y, M, F, V, P, Cit or an analog thereof.
 3. The method of claim 1, wherein at least one of the peptides is a cyclic peptide.
 4. The method of claim 1, wherein the peptide with XG motif and the peptide with XnonG motif are portions of a single peptide.
 5. The method of claim 1, wherein the XnonG peptide is not derived from (pro)fillaggrin, fibrin, fibrinogen, vimentin, cytokeratin 1 or cytokeratin
 9. 6. The method of claim 1, wherein the XG peptide is selected from the group consisting of: 0002-27 H Q K R G Cit G W S R A A 0002-29 H Q R R V Cit G W S R A A 0002-31 H Q R R T Cit G G S R A A 0002-32 H Q R K W Cit G A S R A A 0002-36 H Q F R F Cit G Cit S R A A 0002-37 H Q K W R Cit G R S Cit A A and 0002-63 H Q F R F Cit G W S R A A

and the XnonG peptide is selected from the group consisting of: 0107-32 K P Y T V Cit K F M R R P 0107-35 A R F Q M Cit H Cit R L I R 0107-45 Y S F V W Cit S H A R P R 0113-30 A R F Q M R H Cit R L I R and 0218-36 R N L R L Cit R E R N H A


7. A peptide comprising a sequence with the formula (II): (A1-A2-A3-A4-A5)-Cit-(A6)-(A7-A8-A9-A10-A11)  (II) wherein A1-A2-A3-A4-A5 is an amino acid sequence selected from the group consisting of: RHGRQ IRCitYK HGRQCit GRQCitCit FQMCitH CitWRGM ARFQM QCitYKW KPYTV NRLRL RRRCitY RFKSN RGKSN RWVSQ MKPRY KSFVW YSFVW FQMRH RNMNR and RMGRP

and sequences homologous thereto; A6 represents an amino acid other than glycine; A7-A8-A9-A10-A11 represents an amino acid sequence selected from the group consisting of: KYIIY TNRKF KWCitKI CitRAVI RCitGHS CitGRSR CitYIIY CitRLIR IERKR FMRKP FMRRP ERNHA AVITA TPNRW TYNRW RTPTR RIVVV HARPR RGMCitR and IRFPV

and sequences homologous thereto.
 8. A peptide comprising a sequence with the formula (III): (B1-B2-B3-B4-B5-B6)-Cit-(B7)-(B8-B9-B10-B11)  (III) wherein B1-B2-B3-B4-B5-B6 is an amino acid sequence selected from the group consisting of: INCitRAS ICitKRLY KCitCitYNI RLYFICit IRQGAR CitERCitVQ CitHQRIT RICitRVCit GRNQRY RCitRQHP CitCitRCitVA RPKQHV RKCitGCitR RCitCitRNT RCitQCitFT QLVYLQ QYNRFK CitLRHIR PRCitCitCitK RCitQVRY GRCitHAH ARHVIR RCitGHMF GRNIRV QIFYLCit RQGPIA GVYLVR NCitCitRRV KCitRLCitY GRRCitCitL and RMPHCitH

and sequences homologous thereto; B7 represents an amino acid other than glycine; B8-B9-B10-B11 represents an amino acid sequence selected from the group consisting of: CitHRR CitIRR FRRN AQTT GYPK RPPQ GCitRK PIPR YTIH RIKA CitRVR TRRP TIRP IKCitR RNVV CitRRy CitRPR TRCitCit CitCitGR LCitRCit RVRCit VPRT YCitFR ARCitCit RQCitR HIRR CitMMR CitRICit VRKS PCitCitR and CitRRK

and sequences homologous thereto.
 9. The peptide of claim 7, wherein A6 is selected from the group consisting of Cit, H, I, K, R, S, W, Y, M, F, V and P.
 10. The peptide of claim 7, wherein the peptide is a cyclic peptide comprising a ring of at least 8 amino acids.
 11. The peptide of claim 10, wherein the ring comprises at least 11 amino acids.
 12. The peptide of claim 7 which comprises the peptide sequence R H G R Q Cit Cit K Y I I Y I R Cit Y K Cit I T N R K F R H G R Q Cit Cit Cit Y I I Y A R F Q M Cit H Cit R L I R Q Cit Y K W Cit K I E R K R K P Y T V Cit K F M R K P K P Y T V Cit K F M R R P R N L R L Cit R E R N H A R R R Cit Y Cit R A V I T A R F K S N Cit R T P N R W R G K S N Cit R T Y N R W R F K S N Cit R T Y N R W R G K S N Cit R T P N R W R W V S Q Cit R R T P T R M K P R Y Cit R R I V V V K S F V W Cit S H A R P R Y S F V W Cit S H A R P R R N M N R Cit W R G M Cit R, or R M G R P Cit W I R F P V

or an analog thereof
 13. The peptide of claim 8, which comprises a peptide sequence selected from I N Cit R A S Cit K Cit H R R I Cit K R L Y Cit M Cit I R R K Cit Cit Y N I Cit Cit F R R N R L Y F I Cit Cit R A Q T T I R Q G A R Cit R G Y P K Cit E R Cit V Q Cit R R P P Q Cit H Q R I T Cit V G Cit R K R I Cit R V Cit Cit T P I P R G R N Q R Y Cit L Y T I H R Cit R Q H P Cit H R I K A Cit Cit R Cit V A Cit F Cit R V R R P K Q H V Cit H T R R P R K Cit G Cit R Cit Cit T I R P R Cit Cit R N T Cit H I K Cit R R Cit Q Cit F T Cit Cit R N V V Q L V Y L Q Cit Cit Cit R R Y Q Y N R F K Cit Cit Cit R P R Cit L R H I R Cit Q T R Cit Cit P R Cit Cit Cit K Cit R Cit Cit G R R Cit Q V R Y Cit Cit L Cit R Cit G R Cit H A H Cit P R V R Cit A R H V I R Cit Cit V P R T R Cit G H M F Cit V Y Cit F R G R N I R V Cit Cit A R Cit Cit Q I F Y L Cit Cit H R Q Cit R R Q G P I A Cit L H I R R G V Y L V R Cit L Cit M M R N Cit Cit R R V Cit M Cit R I Cit K Cit R L Cit Y Cit P V R K S G R R Cit Cit L Cit R P Cit Cit R or R M P H Cit H Cit S Cit R R K

or an analog thereof.
 14. A peptide comprising a sequence with the formula (IV) (C1-C2)-(C3-C4-C5)-X-G-C6-(C7-C8-C9-C10)  (IV) wherein C1-C2 is HQ, GF, EG or GV; C3-C4-C5 represents 3 amino acids of which at least 1 is basic, aromatic or V; C6 is a basic or aromatic amino acid, or is A, G, E, P, V, S or Cit or analog thereof; and C7-C8-C9-C10 is SRAA, SCitAA, RPLD, RWE or PGLD; and analogs of the peptide with the formula (IV).
 15. The peptide of claim 14, which is a cyclic peptide comprising a ring of at least 8 amino acids.
 16. The peptide of claim 15, wherein the ring comprises at least 11 amino acids.
 17. The peptide of claim 14, which is H Q R K W Cit G A S R A A H Q H W R Cit G A S R A A H Q F R F Cit G C A S R A A H Q E R R Cit G E S R A A H Q K W R Cit G F S R A A H Q R W K Cit G G S R A A H Q R R T Cit G G S R A A H Q R R G Cit G G S R A A H Q Cit F R Cit G H S R A A G F F S A Cit G H R P L D H Q E R G Cit G K S R A A H Q E K R Cit G K S R A A H Q R W L Cit G K S R A A H Q K R N Cit G K S R A A E G G G V Cit G P R V V E H Q W R H Cit G R S Cit A A H Q K W N Cit G R S R A A H Q K F W Cit G R S R A A H Q K Cit K Cit G R S R A A H Q K W R Cit G R S Cit A A H Q A W R Cit G R S Cit A A H Q N Q W Cit G R S R A A H Q N S K Cit G R S R A A H Q K R R Cit G R S R A A H Q K R F Cit G R S R A A H Q K R Y Cit G R S R A A H Q K R H Cit G R S R A A H Q E R A Cit G S S R A A H Q E K M Cit G V S R A A H Q K R G Cit G W S R A A H Q R R V Cit G W S R A A H Q W N R Cit G W S R A A H Q Q R M Cit G W S R A A H Q S H R Cit G W S R A A H Q F R F Cit G W S R A A H Q K R R Cit G W S R A A or G V K G H Cit G Y P G L D

or an analog thereof.
 18. A peptide comprising at least two linear or cyclic peptide sequences independently selected from the group consisting of: (a) a peptide unit with the formula (II) and (III) and analogs thereof and (b) a peptide with the formula (IV) and analogs thereof.
 19. A diagnostic test kit for determining the presence or absence of autoantibodies to rheumatoid arthritis, comprising a peptide of claim 7, or a mixture thereof, together with at least one further reagent.
 20. A method to select a peptide for diagnosis of RA, which comprises screening a peptide library with antibodies obtained from patients with RA and wherein the peptide library is selected from a group consisting of: Lib(1): H Q E  X   X  Cit  X   X  S R A A

Wherein X=any amino acid except cysteine and tryptophan; Lib(2): H Q  X   X   X  Cit G  X  S R/Cit A A

Wherein X=any amino acid except cysteine but including Citrulline; Lib(3): H Q E  X   X  Cit  X   X  S R/Cit A A

Wherein X=any amino acid except cysteine but including Citrulline; and Lib(4):  X   X   X   X   X   X  Cit  X   X   X   X   X

Wherein X=any amino acid except cysteine but including Citrulline or equivalents thereof.
 21. A peptide obtainable by the method of claim
 20. 22. A diagnostic test kit for determining the presence or absence of autoantibodies to rheumatoid arthritis, comprising a peptide of claim 8, or a mixture thereof, together with at least one further reagent. 